Recently, a number of procedures for determining the base sequence of deoxyribonucleic acid (hereinafter referred to as DNA) directly have been developed. Among others, the chemical sequence determination procedure developed by Maxam and Gilbert is being widely used because it has the advantages of involving relatively simple experimental operations and comparing favorably in rapidity and accuracy with other determination procedures. Details of this procedure are described in Proc. Natl. Acad. Sci., Vol. 74, p. 560, 1977, Tanpakushitsu-Kakusan-Koso [Protein,, Nucleic Acid and Enzyme (Japan)], Vol. 23, p. 182, 1978 and the like. Briefly stated, a DNA sample having one end labeled with radioactive phosphorus is prepared and the four bases constituting the DNA (i.e., guanine, adenine, thymine and cytosine which will hereinafter be referred to as G, A, T and C, respectively) are modified selectively with different chemical reagents. Then, with respect to each base, the DNA strand is cleaved at the position of the modified base and the resulting DNA fragments are subjected to gel electrophoresis in which the fragments are developed and separated in order of molecular chain length. By autoradiography, the developed and separated bands are recorded on an X-ray film and the bases corresponding to the bands on the X-ray film are identified to determine the base sequence of the original DNA sample.
Broadly divided, the above-described procedure can be said to comprise the following four steps:
(1) the step of labeling the 3' or 5'terminus of DNA with radioactive phosphorus; PA1 (2) the step of chemically modifying the labeled DNA in a base-selective manner and cleaving the DNA strand at the position of the modified base; PA1 (3) the step of developing the resulting DNA fragments by gel electrophoresis and recording the bands on an X-ray film; and PA1 (4) the step of analyzing the bands on the X-ray film to determine the base sequence.
This Maxam-Gilbert procedure is being widely used because of its above-described advantages. However, since it has been conventional practice to carry out all of the foregoing steps by hand, determination of the base sequences of many DNA samples requires very troublesome operations and consumes much time. Therefore, it would be desirable to enhance the operating efficiency by mechanization of the steps.
Among the above-described four steps, the present invention is directed to the second step, i.e., the step of chemically modifying the labeled DNA in a base-selective manner and cleaving the DNA strand at the position of the modified base, and is concerned with a method and apparatus for mechanizing this step to enhance the operating efficiency. Conventionally, the second step has been carried out by injecting a sample solution containing the labeled DNA into four tubes made of resin, subjecting them separately to chemical modification reactions specific for four bases (i.e., G, A, T and C) using their respective reagents and reaction conditions, adding ethanol to each tube, cooling the mixture to a temperature of -40.degree. to -70.degree. C., centrifuging it and sucking out the supernatant, washing the precipitate repeatedly (by adding ethanol thereto, centrifuging the mixture and sucking out the supernatant) to remove therefrom any excess reagents used for the chemical modification, adding an aqueous solution of a cleavage reagent (sodium hydroxide or piperidine) to the precipitate and then heating the resulting mixture to cleave the DNA strand at the position of the modified base. The present inventors have concluded that, among the above-described operations, those of adding ethanol to each tube, cooling the mixture and then centrifuging it constitute a difficulty in mechanizing this step. In order to overcome the difficulty, the present inventors have made intensive studies and have found that the adsorption technique can be used in place of the centrifugation one. The present invention has been completed on the basis of this discovery.